|
| Status |
Public on Nov 30, 2009 |
| Title |
continuous light T = 24 hours experiment 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
continuous light T = 24 hours
|
| Organism |
Synechococcus elongatus PCC 7942 = FACHB-805 |
| Characteristics |
experiment: 1
strain: AMC 408
|
| Treatment protocol |
For all experiments, cells are exposed to two light/dark cycles for entrainment prior to release into continuous light, collected at designated times by vacuum filtration, snap frozen in liquid nitrogen, and stored in -80º C. For novobiocin treatment experiment, cells were sampled at T = 56 and T = 64 hours into the circadian cycle. At T = 64 hours, cells were treated with novobiocin sodium salt at concentration 0.1 ug/ml. Cells were then sampled at 5, 10, 30, 90, and 150 minutes using protocol mentioned above.
|
| Growth protocol |
A continuous culture apparatus was developed to keep cells in constant conditions and provide real-time bioluminescence readings. S. elongatus (strain AMC 408) was grown in a 6 liter cylindrical spinner flask (Corning) at a volume of 4.5 liters. Cells were grown in modified BG-11 medium with the following modifications: 0.0010 g/liter of FeNH4 citrate was used instead of 0.0012 g/liter of FeNH4 citrate and citric acid was supplemented at 0.00066 g/liter. Cells were initially inoculated in the presence of antibiotics (5 µg/ml spectinomycin and 5 µg/ml chloramphenicol), and subsequently diluted with modified BG-11 only. Cells were exposed to surface flux of ~25 µmol photons m-2 s-1 white light, bubbled with 500 ml min-1 1% CO2 in air, maintained at 30º C, and stirred at 1 rotation per second. Constant optical density (OD750 0.15) and volume are achieved via a two state controller. OD does not fluctuate more than 8% during an experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from frozen cells in two steps. First, cells were lysed in 65° C phenol/SDS (sodium dodecyl sulfate) by vortexing and the total RNA was purified by phenol/chloroform extraction. Second, total RNA was subjected to DNase I (Promega) treatment followed by a second phenol/chloroform extraction. Total RNA was analyzed on agarose gel and Agilent Bioanalyzer for integrity.
|
| Label |
Cy3
|
| Label protocol |
cDNA was prepared for each individual time-point (foreground channel) as well as for a pool of all time-points (background channel). Spike-in RNA was introduced at different concentrations and ratios to the foreground and background channels prior to reverse transcription in order to ensure proper ratio detection in a wide dynamic range. 5 μg total RNA (plus spike-ins) was reverse-transcribed with random 15-mer primers (Operon) and a 2:3 ratio of amino allyl-UTP:dTTP (Sigma) using SuperScript III reverse-transcriptase (Invitrogen) without amplification. RNA was hydrolyzed and cDNA was purified using Microcon 30 spin column (Millipore). cDNA was labeled with N-Hydroxysuccinimide-ester Cyanine 3 (Cy3, foreground) or Cyanine 5 (Cy5, background) (GE Biosciences) in 0.1 M sodium bicarbonate pH 9.0 for 6 hours. Labeled cDNA was purified (Microcon 30, Millipore) in preparation for hybridization.
|
| |
|
| Channel 2 |
| Source name |
average of samples 1 to 16
|
| Organism |
Synechococcus elongatus PCC 7942 = FACHB-805 |
| Characteristics |
reference: average of samples in experiment 1
|
| Treatment protocol |
For all experiments, cells are exposed to two light/dark cycles for entrainment prior to release into continuous light, collected at designated times by vacuum filtration, snap frozen in liquid nitrogen, and stored in -80º C. For novobiocin treatment experiment, cells were sampled at T = 56 and T = 64 hours into the circadian cycle. At T = 64 hours, cells were treated with novobiocin sodium salt at concentration 0.1 ug/ml. Cells were then sampled at 5, 10, 30, 90, and 150 minutes using protocol mentioned above.
|
| Growth protocol |
A continuous culture apparatus was developed to keep cells in constant conditions and provide real-time bioluminescence readings. S. elongatus (strain AMC 408) was grown in a 6 liter cylindrical spinner flask (Corning) at a volume of 4.5 liters. Cells were grown in modified BG-11 medium with the following modifications: 0.0010 g/liter of FeNH4 citrate was used instead of 0.0012 g/liter of FeNH4 citrate and citric acid was supplemented at 0.00066 g/liter. Cells were initially inoculated in the presence of antibiotics (5 µg/ml spectinomycin and 5 µg/ml chloramphenicol), and subsequently diluted with modified BG-11 only. Cells were exposed to surface flux of ~25 µmol photons m-2 s-1 white light, bubbled with 500 ml min-1 1% CO2 in air, maintained at 30º C, and stirred at 1 rotation per second. Constant optical density (OD750 0.15) and volume are achieved via a two state controller. OD does not fluctuate more than 8% during an experiment.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from frozen cells in two steps. First, cells were lysed in 65° C phenol/SDS (sodium dodecyl sulfate) by vortexing and the total RNA was purified by phenol/chloroform extraction. Second, total RNA was subjected to DNase I (Promega) treatment followed by a second phenol/chloroform extraction. Total RNA was analyzed on agarose gel and Agilent Bioanalyzer for integrity.
|
| Label |
Cy5
|
| Label protocol |
cDNA was prepared for each individual time-point (foreground channel) as well as for a pool of all time-points (background channel). Spike-in RNA was introduced at different concentrations and ratios to the foreground and background channels prior to reverse transcription in order to ensure proper ratio detection in a wide dynamic range. 5 μg total RNA (plus spike-ins) was reverse-transcribed with random 15-mer primers (Operon) and a 2:3 ratio of amino allyl-UTP:dTTP (Sigma) using SuperScript III reverse-transcriptase (Invitrogen) without amplification. RNA was hydrolyzed and cDNA was purified using Microcon 30 spin column (Millipore). cDNA was labeled with N-Hydroxysuccinimide-ester Cyanine 3 (Cy3, foreground) or Cyanine 5 (Cy5, background) (GE Biosciences) in 0.1 M sodium bicarbonate pH 9.0 for 6 hours. Labeled cDNA was purified (Microcon 30, Millipore) in preparation for hybridization.
|
| |
|
| |
| Hybridization protocol |
Each array was hybridized with 150 to 300 ng Cy3 and 150 to 300 ng Cy5 labeled cDNA and rotated (5 rotations min-1) at 60º C for 17 hours in SureHyb chambers (Agilent). Arrays were subsequently washed in 6.7X SSPE and 0.005% N-lauryl sarcosine buffer for at least 1 minute, 0.67X SSPE and 0.005% N-lauryl sarcosine buffer for 1 minute, and then Agilent drying and ozone protection wash for 30 seconds at room temperature (1X SSPE = 0.15 M NaCl, 10 μM sodium phosphate, 1 mM EDTA, pH 7.4).
|
| Scan protocol |
The arrays were immediately scanned using an Axon 4000B scanner at 5 μm resolution.
|
| Description |
no additional information
|
| Data processing |
The average intensity of the Cy3 and Cy5 florescence at each spot was extracted using the GenePix software (Molecular Devices) and loaded into MATLAB (Mathworks). Loess and quantile normalization were performed using the MATLAB bioinformatics toolbox.
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| |
|
| Submission date |
Nov 05, 2009 |
| Last update date |
Nov 30, 2009 |
| Contact name |
Vikram Vijayan |
| E-mail(s) |
vvijayan@fas.harvard.edu
|
| Organization name |
Harvard University
|
| Street address |
52 Oxford Street, Room 440
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
| |
|
| Platform ID |
GPL9534 |
| Series (1) |
| GSE18902 |
Oscillations in supercoiling drive circadian gene expression in cyanobacteria |
|
